ent-Kaurene Biosynthesis in Cell-Free Extracts of Excised Parts of Tall and Dwarf Pea Seedlings

Investigations on the sites of ent-kaur-16-ene (ent-kaurene) biosynthesis were conducted with cell-free extracts from several excised parts of 10-, 13-, and 16-d-old tall and dwarf pea (Pisum sativum L.) seedlings. [¹⁴C]Mevalonic acid was incorporated into ent-kaurene in cell-free extracts from young developing leaves and elongating internodes of tall (`Alaska') and dwarf (`Progress No.9') pea seedlings at all three stages of development. ent-Kaurene biosynthesis also occurred readily in cell-free extracts from shoot tips, petioles, and stipules near the young elongating internodes. The ent-kaurene-synthesizing activity found in young developing tissues declined as tissues matured. Little or no activity was detectable in enzyme extracts from cotyledons and root tips at different stages. In light grown tall pea internodes ent-kaurene-synthesizing activity was low as they began to elongate, reached a maximum when the internodes reached about 2 cm in length and declined as they matured. Activity in extracts of dwarf shoot tips and internodes was generally lower than in equivalent tall plants, but the activity in dwarf leaves and stipules was somewhat higher than in tall plants. With the exception of root tips, there is a strong correlation between growth potential of a tissue and the rate of ent-kaurene biosynthesis in extracts from that tissue.     

Internode Length in Pisum: Gene na May Block Gibberellin Synthesis between ent-7alpha-Hydroxykaurenoic Acid and Gibberellin A(12)-Aldehyde

The elongation response of the gibberellin (GA) deficient genotypes na, ls, and lh of peas (Pisum sativum L.) to a range of GA-precursors was examined. Plants possessing gene na did not respond to precursors in the GA biosynthetic pathway prior to GA₁₂-aldehyde. In contrast, plants possessing lh and ls responded as well as wild-type plants (dwarfed with AMO-1618) to these compounds. The results suggest that GA biosynthesis is blocked prior to ent-kaurene in the lh and ls mutants and between ent-7α-hydroxykaurenoic acid and GA₁₂-aldehyde in the na mutant. Feeds of ent-[³H]kaurenoic acid and [²H]GA₁₂-aldehyde to a range of genotypes supported the above conclusions. The na line WL1766 was shown by gas chromatography-mass spectrometry (GC-MS) to metabolize [²H]GA₁₂-aldehyde to a number of[²H]C₁₉-GAs including GA₁. However, there was no indication in na genotypes for the metabolism of ent-[³H]kaurenoic acid to these GAs. In contrast, the expanding shoot tissue of all Na genotypes examined metabolised ent-[³H]kaurenoic acid to radioactive compounds that co-chromatographed with GA₁, GA₈, GA₂₀, and GA₂₉. However, insufficient material was present for unequivocal identification of the metabolites. The radioactive profiles from HPLC of extracts of the node treated with ent-[³H]kaurenoic acid were similar for both Na and na plants and contained ent-16α,17-dihydroxykaurenoic acid and ent-6α,7α,16β,17-tetrahydroxykaurenoic acid (both characterized by GC-MS), suggesting that the metabolites arose from side branches of the main GA-biosynthetic pathway. Thus, both Na and na plants appear capable of ent-7α-hydroxylation.     

The P450-4 Gene of Gibberella fujikuroi Encodes ent-Kaurene Oxidase in the Gibberellin Biosynthesis Pathway

At least five genes of the gibberellin (GA) biosynthesis pathway are clustered on chromosome 4 of Gibberella fujikuroi; these genes encode the bifunctional ent-copalyl diphosphate synthase/ent-kaurene synthase, a GA-specific geranylgeranyl diphosphate synthase, and three cytochrome P450 monooxygenases. We now describe a fourth cytochrome P450 monooxygenase gene (P450-4). Gas chromatography-mass spectrometry analysis of extracts of mycelia and culture fluid of a P450-4 knockout mutant identified ent-kaurene as the only intermediate of the GA pathway. Incubations with radiolabeled precursors showed that the metabolism of ent-kaurene, ent-kaurenol, and ent-kaurenal was blocked in the transformants, whereas ent-kaurenoic acid was metabolized efficiently to GA₄. The GA-deficient mutant strain SG139, which lacks the 30-kb GA biosynthesis gene cluster, converted ent-kaurene to ent-kaurenoic acid after transformation with P450-4. The B1-41a mutant, described as blocked between ent-kaurenal and ent-kaurenoic acid, was fully complemented by P450-4. There is a single nucleotide difference between the sequence of the B1-41a and wild-type P450-4 alleles at the 3′ consensus sequence of intron 2 in the mutant, resulting in reduced levels of active protein due to a splicing defect in the mutant. These data suggest that P450-4 encodes a multifunctional ent-kaurene oxidase catalyzing all three oxidation steps between ent-kaurene and ent-kaurenoic acid.     

Sites of gibberellin biosynthesis in pea seedlings

Potential sites of gibberellin biosynthesis in 10-day-old `Alaska' pea (Pisum sativum L.) seedlings were investigated using a cell-free ezyme system capable of incorporating [¹⁴C]-mevalonic acid into ent-kaurene. In peas, ent-kaurene is assumed to be a committed intermediate in the gibberellin biosynthetic pathway. Comparative results from enzyme assays using extracts from shoot tips, leaf blades, internodes, and root tips indicate that the highest capacity for ent-kaurene (and presumably gibberellin) synthesis is in those tissues with the greatest potential for growth. The highest rates were obtained with extracts prepared from the fifth (youngest) internode, the fourth (youngest) expanded leaf, and the shoot tip itself. This report represents the first direct evidence that the enzymes responsible for early stages in gibberellin biosynthesis occur in internode tissues with potential for rapid elongation.     

The effect of growth retardants on phytochrome-induced lettuce seed germination

Experiments were carried out to explore the involvement of the plant hormone gibberellin (GA) in the light-induced germination of lettuce seeds. Three growth retardants known to be inhibitors of GA biosynthesis were tested for their effect on red-light-induced germination. Chlormequat chloride (CCC) and AMO-1618 had no effect, but ancymidol was strongly inhibitory. Moreover, the inhibition caused by ancymidol was completely overcome by GA₃. CCC and AMO-1618 inhibit the formation ofent-kaurene, while ancymidol blocks the oxidation ofent-kaurene toent-kaurenoic acid. Ancymidol also was found to inhibit GA-induced dark germination of lettuce seeds, and this inhibition was partially reversed by higher levels of GA. Therefore, the results suggest two possibilities for the relationship between phytochrome and GA in this system: first, the rate-limiting step in the germination of light-sensitive lettuce seeds, that which is regulated by phytochrome, is the oxidation ofent-kaurene toent-kaurenoic acid. Alternatively, red-light treatment may result in the release of active GAlike substances which, in turn, induce germination. In either case the results presented here support the view that phytochrome exerts its effect on lettuce seed germination by means of GA rather than via an independent pathway.     

Kinetics of growth retardant and hormone interactions in affecting cucumber hypocotyl elongation

The capacities of indole-3-acetic acid (IAA) and gibberellin A₃ (GA₃) to counteract the inhibitory effects of (2-chloroethyl) trimethylammonium chloride (CCC), 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride (Amo-1618), and N,N-dimethylaminosuccinamic acid (B-995) on hypocotyl elongation in light-grown cucumber (Cucumis sativus L.) seedlings were investigated. One μg of GA₃ applied to the shoot tip was sufficient to completely nullify the effect of 10 μg of Amo-1618 or 25 μg of B-995 applied simultaneously to the shoot tip, and 10 μg of GA₃ completely counteracted the effect of 10⁻³ m CCC added to the root medium. One μg of IAA counteracted the effect of 10⁻³ m CCC in the root medium, but IAA did not nullify the action of either Amo-1618 or B-995. Experiments were conducted using 2 growth retardants simultaneously, which indicated that Amo-1618 and CCC inhibit a common process, namely GA biosynthesis, essential to hypocotyl elongation. However, since the effect of CCC was overcome by applications of both GA and IAA, growth retardation resulting from treatment with CCC apparently is not due solely to inhibition of GA biosynthesis. B-995 did not interact additively with either Amo-1618 or CCC, which suggests that B-995 affects a process different from those affected by the other 2 retardants. Thus, while inhibition evoked by B-995 is reversible by applied GA, the action of B-995 does not appear to be inhibition of GA biosynthesis.     

Arabidopsis ent-Kaurene Oxidase Catalyzes Three Steps of Gibberellin Biosynthesis

The Arabidopsis GA3 cDNA was expressed in yeast (Saccharomyces cerevisiae) and the ability of the transformed yeast cells to metabolize ent-kaurene was tested. We show by full-scan gas chromatography-mass spectrometry that the transformed cells produce ent-kaurenoic acid, and demonstrate that the single enzyme GA3 (ent-kaurene oxidase) catalyzes the three steps of gibberellin biosynthesis from ent-kaurene to ent-kaurenoic acid.     

Relationship between Chloroplast Development and ent-Kaurene Biosynthesis in Peas

Treatment of etiolated pea (Pisum sativum (L. cv. Alaska) seedlings with 2′-isopropyl-4′-(trimethylammonium chloride)-5′-methylphenyl piperidine-1-carboxylate (Amo-1618) prior to irradiation with white light inhibits photomorphogenesis and formation and stacking of thylakoid membranes in the chloroplasts, as well as (−)-kaur-16-ene (ent-kaurene)biosynthesis. Exogenous gibberellic acid also inhibits greening. A crudely determined action spectrum for the photoinduction of ent-kaurene biosynthesis shows two peaks, one in the blue region at 458 to 490 nanometers and another in the red region at 606 to 678 nanometers. The possible participation of phytochrome in the photoinduction of ent-kaurene biosynthesis is indicated by comparative effects of red, far red, and alternating red/far red irradiations on enhancement of enzyme activity. The activity of blue light as well as red shows a similarity of the photoinduction of ent-kaurene synthesis activity to the high irradiance responses, and indicates probable participation of a second photoreceptor. From these observations, it is concluded that photoinduction of ent-kaurene biosynthesis and chloroplast development in shoots are closely linked processes.     

Induction ofent-kaurene biosynthesis by low temperature in dwarf peas

Germinating pea (Pisum sativum L.) seeds of two dwarf cultivars, “Progress No. 9” and “Green Arrow”, and two tall cultivars, “Alaska” and “Alderman”, were treated with low temperature (3–5°C) for 14 days and then transferred to normal growing conditions (19–21°C for 16 h/14.5–16.5°C for 8 h) for an additional 10 days. Biosynthesis of [¹⁴C]ent-kaurene from [¹⁴C]2-mevalonic acid (2-MVA) was assayed in cell-free enzyme extracts prepared from shoot tips 10 days after cold treatment and was compared with activity in enzyme extracts prepared from noncold-treated, 10-day-old control plants. Shoot lengths of cold-treated plants were measured throughout a 35-day period and compared with shoot lengths of plants grown without cold treatment for 25–35 days. Low temperature induced a five-to 10-fold enhancement ofent-kaurene, hence potentially gibberellin (GA), biosynthesis in seedlings of the two dwarf cultivars but not in the tall cultivars. However, the lack of an increase in growth rate in the cold-treated dwarfs indicated that endogenous GA biosynthesis remained blocked at some point beyondent-kaurene in the biosynthetic pathway. Since the late-flowering “Alderman” cultivar did not exhibit enhanced biosynthesis ofent-kaurene, it appears that if vernalization in late-flowering cultivars of peas is correlated with enhanced GA biosynthesis, it is not the early part of the biosynthetic pathway which is affected.     

Inhibition of Gibberellin Production in the Fungi Gibberella fujikuroi and Sphaceloma manihoticola by Plant Growth Retardants

The effect of different types of plant growth retardants on fungal gibberellin (GA) formation has been studied in cultures of Gibberella fujikuroi and Sphaceloma manihoticola. Quaternary ammonium compounds (chlormequat chloride, mepiquat chloride, Amo-1618), triazoles (uniconazole and several experimental compounds), and the norbornanodiazetine tetcyclacis inhibited GA biosynthesis in both fungal species. Concentrations between 2 × 10⁻⁴ and 10⁻⁹m were required for a 50% inhibition of the production of gibberellin A₃ in Gibberella fujikuroi and of giberellin A₄ in Sphaceloma manihoticola. The formation of other prominent GAs was affected at a similar degree of intensity. Tetcyclacis was the most active compound in both fungi. Compared to the growth retardants mentioned above, the biological activity of chlorphonium chloride was low. The acylcyclohexanediones prohexadione and LAB 198 999 had virtually no activity. Most likely, this lack of activity is due to a rapid metabolism of the compounds in the cultures. For the triazole-type compounds and tetcyclacis, a relatively distinct correlation exists in their ability to inhibit GA formation in fungal cultures, to block ent-kaurene oxygenase in a cell-free system, and to reduce shoot growth of rice seedlings. Due to differences in their metabolic fate and species specificities, such conclusions cannot be made for the other compounds.     

Selection for low dormancy in annual ryegrass (Lolium rigidum) seeds results in high constitutive expression of a glucose-responsive α-amylase isoform

Background and Aims

α-Amylase in grass caryopses (seeds) is usually expressed upon commencement of germination and is rarely seen in dry, mature seeds. A heat-stable α-amylase activity was unexpectedly selected for expression in dry annual ryegrass (Lolium rigidum) seeds during targeted selection for low primary dormancy. The aim of this study was to characterize this constitutive activity biochemically and determine if its presence conferred insensitivity to the germination inhibitors abscisic acid and benzoxazolinone.

Methods

α-Amylase activity in developing, mature and germinating seeds from the selected (low-dormancy) and a field-collected (dormant) population was characterized by native activity PAGE. The response of seed germination and α-amylase activity to abscisic acid and benzoxazolinone was assessed. Using an alginate affinity matrix, α-amylase was purified from dry and germinating seeds for analysis of its enzymatic properties.

Key Results

The constitutive α-amylase activity appeared late during seed development and was mainly localized in the aleurone; in germinating seeds, this activity was responsive to both glucose and gibberellin. It migrated differently on native PAGE compared with the major activities in germinating seeds of the dormant population, but the enzymatic properties of α-amylase purified from the low-dormancy and dormant seeds were largely indistinguishable. Seed imbibition on benzoxazolinone had little effect on the low-dormancy seeds but greatly inhibited germination and α-amylase activity in the dormant population.

Conclusions

The constitutive α-amylase activity in annual ryegrass seeds selected for low dormancy is electrophoretically different from that in germinating seeds and its presence confers insensitivity to benzoxazolinone. The concurrent selection of low dormancy and constitutive α-amylase activity may help to enhance seedling establishment under competitive conditions.     

The Multiple Forms of alpha-Amylase Enzyme of the Araucaria Species of South America: A. araucana (Mol.) Koch and A. angustifolia (Bert.) O. Kutz : A Comparative Study

α-Amylase is one of the major enzymes present in the seeds of both Araucaria species of South America and it initiates starch hydrolysis during germination and early seedling growth. The pattern of the multiple forms of α-amylase of the two Araucaria species was investigated by electrophoresis and isoelectrofocusing of the native enzyme in polyacrylamide gels. The enzyme forms were compared in the embryo and megagametophyte of quiescent seeds and of seeds imbibed for 18, 48, and 90 hours. Specific α-amylase enzyme forms appear and disappear during these imbibition periods showing both similarities and differences between tissues and species. Before imbibition, there are five α-amylase forms identical in both tissues, but different between species. After 18 hours of imbibition, there are two enzyme forms in both tissues of Araucaria araucana seeds, only one form in the embryo of Araucaria angustifolia but two forms in the megagametophyte of this specie. After 48 hours of seed imbibition, most of the enzyme forms present in quiescent seeds reappear. At 90 hours of imbibition different enzyme forms are detected in the embryo with respect to the gametophyte. The changes in form patterns of α-amylase are discussed according to a possible regulation of gene expression by endogenous gibberellins.     

Control of alpha-Amylase Development in Cotyledons during and following Germination of Mung Bean Seeds

Developmental patterns of α-amylase in Vigna radiata cotyledons during and following germination were quite different depending on the differences in the treatments of cotyledons during the imbibitional stage. When axis-detached cotyledons were imbibed in water with seed-coats attached, α-amylase activity did not increase and remained low. On the other hand, when the cotyledons were imbibed in water after seed-coat removal, the enzyme activity increased markedly. If the axis was attached to the cotyledons, α-amylase showed a marked development even under the former imbibition conditions. These changes in the enzyme activity were in parallel with those in the enzyme content, and the content, in turn, was dependent upon the availability of mRNA for α-amylase. We propose that the regulation of the development of α-amylase in cotyledons may involve some factor(s) inhibitory to accumulation of α-amylase mRNA, which is present in dry cotyledons and can be removed from cotyledons by leakage or by the presence of the axis.     

No Effect of 5-Fluorouracil on the Properties of Purified alpha-Amylase from Barley Half-seeds

α-Amylase has been purified from de-embryonated seeds of barley (Hordeum vulgare L. cv. Betzes) which have been incubated on 10⁻⁶ m gibberellic acid (GA₃) following 3 days of imbibition in buffer. Incubation of the half-seeds in up to 10⁻² m 5-fluorouracil (5-FU) during the entire incubation period, including imbibition, had no effect on any of the following characteristics of purified α-amylase: thermal stability in the absence of calcium, molecular weight of the enzyme, isozyme composition, specific activity, or the amount of α-amylase synthesized by the aleurone tissue. The synthesis of rRNA and tRNA was strongly inhibited by 5-FU, indicating that the analog had entered the aleurone cells. These results are not in agreement with those of Carlson (Nature New Biology 237: 39-41 [1972]) who found that treatment of barley aleurone with 10⁻⁴ m 5-FU prior to the addition of GA₃ resulted in decreased thermal stability of GA₃-induced α-amylase and who interpreted this as evidence that the mRNA for α-amylase was synthesized during the imbibition of the aleurone tissue and independently of gibberellin action. Results of the present experiments indicate that the thermal stability of highly purified α-amylase is not altered by treatment of barley half-seeds with 5-FU, and that 5-FU cannot be used as a probe to examine the timing of α-amylase mRNA synthesis.     

Metabolism of ent-Kaurene to Gibberellin A(12)-Aldehyde in Young Shoots of Normal Maize

Young shoots of normal maize (Zea mays L.) were used to determine both the stepwise metabolism of ent-kaurene to gibberellin A₁₂-aldehyde and the endogenous presence of the members in this series. Each of the five steps in the sequence was established by feeds of 17-¹³C, ³H-labeled kauranoids to cubes from the cortex of elongating internodes, to homogenates from the cortex of elongating internodes, and/or to homogenates from dark-grown seedlings. The ¹³C-metabolites were identified by Kovats retention indices (KRI) and full-scan capillary gas chromatography-mass spectrometry (GC-MS). Five substrates and the final product in this sequence were shown to be native by the isotopic dilution of 17-¹³C, ³H-labeled substrates added as internal standards to extracts obtained from elongating internodes. Evidence for the isotopic dilution was obtained by KRI and full-scan capillary GC-MS. Thus, we document the presence in young maize shoots of the metabolic steps, ent-kaurene → ent-kaurenol → ent-kaurenal → ent-kaurenoic acid → ent-7 α-hydroxykaurenoic acid → gibberellin A₁₂-aldehyde.     

Biosynthesis of ent-kaurene in cell-free extracts of Pisum sativum shoot tips

Soluble enzyme preparations from pea shoot tips incorporated mevalonic acid-2-¹⁴C into ent-kaurene-¹⁴C, squalene-¹⁴C and other products. The assay for either ent-kaurene or squalene is quite direct; both products can be obtained apparently free of radioactive contaminants by TLC on silica gel G in hexane. The enzyme system is dependent upon added ATP and Mn²⁺ or Mg²⁺, with Mn²⁺ being a more effective activator than Mg²⁺ under the experimental conditions. Reduced pyridine nucleotide had no effect on ent-kaurene production but stimulated squalene synthesis. The accumulation of both ent-kaurene and squalene was stimulated by dithiothreitol and carbon monoxide and was reduced by the addition of particulate cell components. AMO-1618 inhibited ent-kaurene production and had no effect on the synthesis of squalene. Enzyme extracts from shoot tips are much less active in ent-kaurene synthesis than extracts from the cotyledons of immature seeds on either a fresh weight or protein basis.     

beta-Amylases in Cereals : A Study of the Maize beta-Amylase System

β-Amylase of maize (Zea mays L.) caryopses was studied during development and germination by means of enzymic, electrophoretic, and immunochemical techniques. β-Amylase activity increased during caryopsis development to a maximum value at the beginning of the water content plateau (at this stage the enzyme was located primarily within the pericarp) and then decreased. Almost no β-amylase (activity or antigen) was found in either free or bound forms in the mature maize caryopsis. The activity increased again during seedling growth and reached much higher values. Both the aleurone layer (to a major extent) and the scutellum produced and secreted β-amylase during germination, the secretion being stimulated by Ca²⁺. No posttranslational modification of the enzyme was detected during germination. The molecular specific activity of the enzyme remained unchanged during the observed periods, indicating that the regulation of the activity is based essentially on protein turnover. The enzyme from developing and germinating caryopses was found to be identical in terms of antigenicity, isoelectric point, and molecular mass to the β-amylases extracted from the roots and the leaves of the maize seedling. The maize β-amylase resembles in all respects the ubiquitous β-amylase described for rye and wheat, whereas the major β-amylase of those cereals appears to be lacking in the maize caryopsis.     

CYP701A26 is characterized as an <Emphasis Type="Italic">ent</Emphasis>-kaurene oxidase with putative involvement in maize gibberellin biosynthesis

Objectives

To characterize the ent-kaurene oxidase (KO) involved in maize (Zea mays) gibberellin (GA) biosynthesis.

Results

Two putative KO genes were identified in maize based on the homologous alignment. Biochemical characterization indicated that one of them encoded a cytochrome P450 monooxygenase (P450) CYP701A26, which reacted with ent-kaurene to form ent-kaurenoic acid, the key intermediate of GA biosynthesis. CYP701A26 showed constitutive expression in active growing tissues and no inducible expression, which led to putative designation of CYP701A26 as the ZmKO. CYP701A26 exhibited substrate promiscuity to catalyze oxidation of other labdane related diterpenes. Another maize KO homologue, CYP701A43 did not show any catalytic activities on ent-kaurene or other tested diterpenes. It exhibited inducible gene expression and might accept unknown substrates to play roles in specialized metabolism for stress response.

Conclusions

CYP701A26 was characterized to exhibit ent-kaurene oxidase activity with substrate promiscuity and might be involved in maize GA biosynthesis, and its homologue CYP701A43 did not show such function and might play roles in stress response.

     

The GA2 Locus of Arabidopsis thaliana Encodes ent-Kaurene Synthase of Gibberellin Biosynthesis

The ga2 mutant of Arabidopsis thaliana is a gibberellin-deficient dwarf. Previous biochemical studies have suggested that the ga2 mutant is impaired in the conversion of copalyl diphosphate to ent-kaurene, which is catalyzed by ent-kaurene synthase (KS). Overexpression of the previously isolated KS cDNA from pumpkin (Cucurbita maxima) (CmKS) in the ga2 mutant was able to complement the mutant phenotype. A genomic clone coding for KS, AtKS, was isolated from A. thaliana using CmKS cDNA as a heterologous probe. The corresponding A. thaliana cDNA was isolated and expressed in Escherichia coli as a fusion protein. The fusion protein showed enzymatic activity that converted [³H]copalyl diphosphate to [³H]ent-kaurene. The recombinant AtKS protein derived from the ga2–1 mutant is truncated by 14 kD at the C-terminal end and does not contain significant KS activity in vitro. Sequence analysis revealed that a C-2099 to T base substitution, which converts Gln-678 codon to a stop codon, is present in the AtKS cDNA from the ga2–1 mutant. Taken together, our results show that the GA2 locus encodes KS.